Stable metal/conductive polymer composite colloids and methods for making and using the same

ABSTRACT

Stable metal/conductive polymer composite colloids and methods for making the same are provided. The subject colloids find use in a variety of different applications, including analyte detection applications. Also provided are kits that include the subject colloids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 60/621,258 filed on Oct. 21, 2004; thedisclosure of which is herein incorporated by reference.

INTRODUCTION Background of the Invention

Nanoparticles of noble metals such as gold or silver can be prepared invarious geometrical forms such as spheres, rods or pyramids. These smallobjects contain the metallic element in chemically reduced form anddepending on the way they are prepared, they can be either stored asreduced powdered solids, or held in stable suspension in solvents suchas water or various organic solvents (i.e., as a colloid). Because ofthe nanometer size of the particles, the naked eye cannot distinguishsuch suspensions from true solutions, although the microscope can, andsuch suspensions are therefore termed colloidal solutions. The particlesare therefore easily cast on various supports to form well-definedcircuits.

The study of these metal nanoparticles has been an extremely active areain recent years because of their unique electronic, optical andcatalytic properties. Since light is associated with an electromagneticfield, the opto-electronic properties of the nanoparticles areparticularly interesting. Indeed, because they are metallic and capableof conducting electricity, the noble metal nanoparticles are surroundedat their surface by a dense cloud of conducting electrons. When theseelectrons are excited by light, the electromagnetic radiation combineswith these electrons to form collective oscillations that radiate awayfrom the particle surface. As a result, the particles exhibit specificlight absorption, reflection, emission and scattering properties thatcan be successfully applied in various fields such as analyte detection,electron transport or information storage. Most of the particles studiedso far are homogeneously made of the same metal. However, it has beenshown recently that it was possible to construct nano-objects made ofdifferent metals alternating in the structure. This new advance opensthe way to their use as nano bar-codes in numerous applications.

Because of the field acknowledge potential of nanoparticles in a varietyof diverse applications, there is continued interest in the developmentof new types of nanoparticles and applications therefore.

SUMMARY OF THE INVENTION

Stable metal/conductive polymer composite colloids and methods formaking the same are provided. The subject colloids find use in a varietyof different applications, including analyte detection applications.Also provided are kits that include the subject colloids.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. GP-HPLC profile of poly(thiophene-3-carboxylic acid) [left] andof PANI-COOH [right] solutions. The elution time of bovine serum albumin(BSA) is indicated (arrow) for reference.

FIG. 2. Stabilization of colloidal gold nanoparticles toward saltflocculation by PANI-COOH at different pH values.

FIG. 3. Stabilization of colloidal gold nanoparticles towardflocculation by salt by increasing concentrations of PANI-COOH at fixedpH. Original absorption wavelengths of PANI-COOH and gold nanoparticlesare respectively shown by up and down open triangles.

FIG. 4. Progressive stabilization of gold nanoparticles towardflocculation by salt by increasing concentrations of PANI-COOH at fixedpH, as measured by the O.D. at the maximal wavelength after saltaddition.

FIG. 5. Visible absorption spectra of solutions of PANI-COOH, colloidalgold nanoparticles and PANI-COOH-gold composite.

FIG. 6. Absorption spectra of the composite material buffered at pH 9taken immediately after synthesis (solid line) and after three and ahalf months of storage at room temperature (dashed line).

FIG. 7. Difference spectra between pH 4.95 and pH 9 of the compositematerial (solid line) and of the PANI-COOH solution (dashed line).

FIG. 8. Dose-response curves observed respectively with PANI-COOH (opencircles) or colloidal nanocomposite (closed circles) solutions afterreaction with increasing does of ascorbic acid. Data are averages±S.D.of triplicate measurements.

FIG. 9. Effect of glycerol on gold nanocolloids as measured bydifference spectroscopy.

FIG. 10. Effect of glycerol on PANI-COOH-gold composite nanocolloids asmeasured by difference spectroscopy.

FIG. 11. Evolution of absorbances at typical wavelengths ofPANI-COOH-gold composite nanoparticles as a function of changes in therefractive index of the medium.

FIG. 12. Comparison of the changes in absorbance at 350 nm for thecomposite material and changes at 575 nm for gold nanoparticles as afunction of the change in refractive index of the medium.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Still, certain elements aredefined below for the sake of clarity and ease of reference.

The term “colloid” refers to a fluid composition of microscopicparticles suspended in a liquid medium. In representative colloids, theparticles therein are between one nanometer and one micrometer in size.

The term metal colloid refers to a colloid in which the suspendedmicroscopic particles are metal particles.

The term “noble metal” refers to Group VIII metals of the Periodic Tableincluding, but not limited to: platinum, iridium, palladium and thelike, as well as gold, silver etc.

The term “conductive polymer” means an electrically conductive polymericmaterial. In representative embodiments, conductive polymers are organicpolymers, such as p-conjugated organic polymers. For example, employedmay be polypyrroles such as polypyrrole, poly(N-substituted pyrrole),poly(3-substituted pyrrole), and poly(3,4-disubstituted pyrrole);polythiophenes such as polythiophene, poly(3-substituted thiophene),poly(3,4-disubstituted thiophene), and polybenzothiophene;polyisothianaphthenes such as polyisothianaphthene;polythienylenevinylenes such as polythienylenevinylene;poly(p-phenylenevinylenes) such as poly(p-phenylenevinylene);polyanilines such as polyaniline, poly(N-substituted aniline),poly(3-substituted aniline), and poly(2,3-substituted aniline);polyacetylenes such as polyacetylene; polydiacetylenes such aspolydiacetylene; polyazulenes such as polyazulene; polypyrenes such aspolypyrene; polycarbazoles such as polycarbazole and poly(N-substitutedcarbazole), polyselenophenes such as polyselenophene; polyfurans such aspolyfuran and polybenzofuran; poly(p-phenylens) such aspoly(p-phenylene); polyindoles such as polyindole; polypyridazines suchas polypyridazine; polyacenes such as naphthacene, pentacene, hexacene,heptacene, dibenzopentacene, tertabenzopentacene, pyrene, dibenzopyrene,chrysene, perylene, coronene, Terylene, ovalene, quoterylene, andcircumanthracene; derivatives (such as triphenodioxazine,triphenodithiazine, hexacene-6,15-quinone) which are prepared bysubstituting some of carbon atoms of polyacens with atoms such as N, S,and O, or a functional group such as a carbonyl group; polymers such aspolyvinylcarbazoles, polyphenylenesulfide, and polyvinylenesulfide. Ofparticular interest in representative embodiments are polypyrrole ,polythiophene, polyaniline or their derivatives.

As is known in the art, the conducting polymer may be doped byincorporating into the polymer materials having a functional group suchas a dimethylamino group, a cyano group, a carboxyl group and a nitrogroup, materials such as benzoquinone derivatives, andtetracyanoethylene as well as tetracyanoquinodimethane, and derivativesthereof, which work as an acceptor which accepts electrons, or, forexample, materials having a functional group such as an amino group, atriphenyl group, an alkyl group, a hydroxyl group, an alkoxy group, anda phenyl group; substituted amines such as phenylenediamine; anthracene,benzoanthracene, substituted benzoanthracenes, pyrene, substitutedpyrene, carbazole and derivatives thereof, and tetrathiafulvalene andderivatives thereof, which work as a donor which is an electron donor.The doping, as described herein, means that electron accepting molecules(acceptors) or electron donating molecules (donors) are incorporated insaid thin film employing doping. Employed as dopants used in the presentinvention may be either acceptors or donors

The term “metal/conductive polymer composite colloid” refers to acolloid made up of metal particles having a conductive polymer presenton a surface thereof.

The term “adsorb” refers to the adhesion in an extremely thin layer ofmolecules (e.g., water-soluble polymer molecules) to the surfaces ofsolid bodies, e.g., metal particles, with which they are in contact.

A material is “water-soluble” if it dissolves in water. With respect tothe conductive polymers of the present invention, such are consideredwater-soluble if at least about 0.02 g will dissolve in at least about100 ml water at standard temperature and pressure (STP) conditions.

As used herein, the term “contacting” means to bring or put together. Assuch, a first item is contacted with a second item when the two itemsare brought or put together, e.g., by touching them to each other. Theterm “combining” refers to contacting two different compositions in amanner such that they become a single composition.

The term “agitation” refers to application of physical movement to acomposition, such that the components thereof move relative to eachother. As such, the term agitation is employed broadly to refer tomixing, stirring, and the like.

The term “ligand” as used herein refers to any type of molecule that isa member of a specific binding pair. Ligands of interest include, butare not limited to biomolecules, where the term “biomolecule” means anyorganic or biochemical molecule, group or species of interest, e.g.,that can specifically bind to an analyte of interest. Exemplarybiomolecules include peptides, proteins, amino acids and nucleic acids,small organic and inorganic molecules, etc.

The term “peptide” as used herein refers to any compound produced byamide formation between a carboxyl group of one amino acid and an aminogroup of another group.

The term “oligopeptide” as used herein refers to peptides with fewerthan about 10 to 20 residues, i.e. amino acid monomeric units.

The term “polypeptide” as used herein refers to peptides with more thanabout 10 to about 20 residues. The terms “polypeptide” and “protein” maybe used interchangeably.

The term “protein” as used herein refers to polypeptides of specificsequence of more than about 50 residue and includes D and L forms,modified forms, etc.

The term “nucleic acid” as used herein means a polymer composed ofnucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compoundsproduced synthetically (e.g., PNA as described in U.S. Pat. No.5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions.

The terms “nucleoside” and “nucleotide” are intended to include thosemoieties that contain not only the known purine and pyrimidine basemoieties, but also other heterocyclic base moieties that have beenmodified. Such modifications include methylated purines or pyrimidines,acylated purines or pyrimidines, or other heterocycles. In addition, theterms “nucleoside” and “nucleotide” include those moieties that containnot only conventional ribose and deoxyribose sugars, but other sugars aswell. Modified nucleosides or nucleotides also include modifications onthe sugar moiety, e.g., wherein one or more of the hydroxyl groups arereplaced with halogen atoms or aliphatic groups, or are functionalizedas ethers, amines, or the like.

Also of interest are small organic and inorganic molecules. For example,organic molecules, such small organic compounds having a molecularweight of more than 50 and less than about 2,500 daltons are of interestas ligands in certain embodiments. Small organic compounds may includefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two of thefunctional chemical groups. Such compounds may include cyclical carbonor heterocyclic structures and/or aromatic or polyaromatic structuressubstituted with one or more of the above functional groups.

In certain embodiments, a linking group is employed to indirectly bind aligand to a surface of a composite nanoparticle. Where linking groupsare employed, such groups are chosen to provide for covalent attachmentof the ligand moiety and the surface through the linking group. Linkinggroups of interest may vary widely depending on the nature of the targetand blocking ligand moieties. A variety of linking groups are known tothose of skill in the art and find use in the subject bifunctionalmolecules. Generally, such linkers will comprise a spacer groupterminated at either end with a reactive functionality capable ofcovalently bonding to the ligand or surface. Spacer groups of interestpossibly include aliphatic and unsaturated hydrocarbon chains, spacerscontaining heteroatoms such as oxygen (ethers such as polyethyleneglycol) or nitrogen (polyamines), peptides, carbohydrates, cyclic oracyclic systems that may possibly contain heteroatoms. Spacer groups mayalso be comprised of ligands that bind to metals such that the presenceof a metal ion coordinates two or more ligands to form a complex.Specific spacer elements include: 1,4-diaminohexane, xylylenediamine,terephthalic acid, 3,6-dioxaoctanedioic acid,ethylenediamine-N,N-diacetic acid,1,1′-ethylenebis(5-oxo-3-pyrrolidinecarboxylic acid),4,4′-ethylenedipiperidine. Potential reactive functionalities includenucleophilic functional groups (amines, alcohols, thiols, hydrazides),electrophilic functional groups (aldehydes, esters, vinyl ketones,epoxides, isocyanates, maleimides), functional groups capable ofcycloaddition reactions, forming disulfide bonds, or binding to metals.Specific examples include primary and secondary amines, hydroxamicacids, N-hydroxysuccinimidyl esters, N-hydroxysuccinimidyl carbonates,oxycarbonylimidazoles, nitrophenylesters, trifluoroethyl esters,glycidyl ethers, vinylsulfones, and maleimides. Specific linker groupsthat may find use in the subject bifunctional molecules includeheterofunctional compounds, such as azidobenzoyl hydrazide,N-[4-(p-azidosalicylamino)butyl]-3′-[2′-pyridyidithio]propionamid),bis-sulfosuccinimidyl suberate, dimethyladipimidate,disuccinimidyltartrate, N--maleimidobutyryloxysuccinimide ester,N-hydroxy sulfosuccinimidyl 4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3′-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate,3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP),4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimideester (SMCC), and the like.

The terms “ribonucleic acid” and “RNA” as used herein refer to a polymercomposed of ribonucleotides.

By “homogenous” is meant that a composition is of the same or a similarkind or nature throughout, i.e., of uniform structure or compositionthroughout.

The terms “deoxyribonucleic acid” and “DNA” as used herein mean apolymer composed of deoxyribonucleotides.

The term “oligonucleotide” as used herein denotes single strandednucleotide multimers of from about 10 to 100 nucleotides and up to 200nucleotides in length.

A “biopolymer” is a polymer of one or more types of repeating units.Biopolymers are typically found in biological systems (although they maybe made synthetically) and may include peptides or polynucleotides, aswell as such compounds composed of or containing amino acid analogs ornon-amino acid groups, or nucleotide analogs or non-nucleotide groups.This includes polynucleotides in which the conventional backbone hasbeen replaced with a non-naturally occurring or synthetic backbone, andnucleic acids (or synthetic or naturally occurring analogs) in which oneor more of the conventional bases has been replaced with a group(natural or synthetic) capable of participating in Watson-Crick typehydrogen bonding interactions. Polynucleotides include single ormultiple stranded configurations, where one or more of the strands mayor may not be completely aligned with another. For example, a“biopolymer” may include DNA (including cDNA), RNA, oligonucleotides,and PNA and other polynucleotides as described in U.S. Pat. No.5,948,902 and references cited therein (all of which are incorporatedherein by reference), regardless of the source.

The phrase “optical property” refers to an optical parameter, i.e., aproperty whose value determines the characteristic or behavior ofsomething, where representative optical properties include, but are notlimited to: light absorption, light emission, light reflection and lightscattering.

The terms “reference” and “control” are used interchangeably to refer toa known value or set of known values against which an observed value maybe compared. As used herein, known means that the value represents anunderstood parameter, e.g., light absorption, light emission, etc.

The term “assessing” and “evaluating” are used interchangeably to referto any form of measurement, and includes determining if an element ispresent or not. The terms “determining,” “measuring,” “assessing,” and“assaying” are used interchangeably and include both quantitative andqualitative determinations. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

As used herein, the term “detecting” means to ascertain a signal, eitherqualitatively or quantitatively.

The term “binding” refers to two objects associating with each other toproduce a stable composite structure. In certain embodiments, bindingbetween two complementary nucleic acids may be referred to asspecifically hybridizing. The terms “specifically hybridizing,”“hybridizing specifically to” and “specific hybridization” and“selectively hybridize to,” are used interchangeably and refer to thebinding, duplexing, or hybridizing of a nucleic acid moleculepreferentially to a particular nucleotide sequence under stringentconditions.

The term “screening” refers to determining the presence of something ofinterest, e.g., an analyte, an occurrence, etc. As used herein, the term“determining” means to identify, i.e., establishing, ascertaining,evaluating or measuring, a value for a particular parameter of interest,e.g., a hybridization parameter. The determination of the value may bequalitative (e.g., presence or absence) or quantitative, where aquantitative determination may be either relative (i.e., a value whoseunits are relative to a control (i.e., reference value) or absolute(e.g., where a number of actual molecules is determined).

The term “sample” as used herein refers to a fluid composition, where incertain embodiments the fluid composition is an aqueous composition.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Stable metal/conductive polymer composite colloids and methods formaking the same are provided. The subject colloids find use in a varietyof different applications, including analyte detection applications.Also provided are kits that include the subject colloids.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

As summarized above, the subject invention provides stablemetal/conductive polymer composite colloids and methods for making andusing the same. In further describing the subject invention,representative embodiments of the subject colloids are reviewed first ingreater detail, followed by a discussion of representative fabricationprotocols and methods for using the subject colloids. In addition, areview of representative kits that include the subject colloids isprovided.

Metal/Conductive Polymer Composite Colloids

As summarized above, the present invention provides metal/conductivepolymer composite colloids. A feature of the subject colloids is thatthey are stable. As used herein, the term stable refers to the abilityof the particles of the colloid to remain in suspension in the carriermedium of the colloid, e.g., the particles do not precipitate out ofsuspension to any significant extent. With respect to the subjectcolloids, the colloids are stable when maintained at STP conditions fora period of time that is at least about 1 month long, such as at leastabout 3 months long, including at least about 6 months long, and are, inrepresentative embodiments, stable for periods of up to one year orlonger, such as up to two years or longer, including up to five years orlonger.

As the subject colloids are composites of metals and conductingpolymers, they include both a metal component and a conducting polymercomponent. The metal component of the subject colloids is, inrepresentative embodiments, a noble metal. As indicated above, noblemetals of interest include, but are not limited to: Group Vil metals ofthe Periodic Table including, but not limited to: platinum, iridium,palladium and the like, as well as gold, silver etc.

The term “conductive polymer” means an electrically conductive polymericmaterial. In representative embodiments, conductive polymers are organicpolymers, such as p-conjugated organic polymers. For example, employedmay be polypyrroles such as polypyrrole, poly(N-substituted pyrrole),poly(3-substituted pyrrole), and poly(3,4-disubstituted pyrrole);polythiophenes such as polythiophene, poly(3-substituted thiophene),poly(3,4-disubstituted thiophene), and polybenzothiophene;polyisothianaphthenes such as polyisothianaphthene;polythienylenevinylenes such as polythienylenevinylene;poly(p-phenylenevinylenes) such as poly(p-phenylenevinylene);polyanilines such as polyaniline, poly(N-substituted aniline),poly(3-substituted aniline), and poly(2,3-substituted aniline);polyacetylnenes such as polyacetylene; polydiacetylens such aspolydiacetylene; polyazulenes such as polyazulene; polypyrenes such aspolypyrene; polycarbazoles such as polycarbazole and poly(N-substitutedcarbazole), polyselenophenes such as polyselenophene; polyfurans such aspolyfuran and polybenzofuran; poly(p-phenylens) such aspoly(p-phenylene); polyindoles such as polyindole; polypyridazines suchas polypyridazine; polyacenes such as naphthacene, pentacene, hexacene,heptacene, dibenzopentacene, tertabenzopentacene, pyrene, dibenzopyrene,chrysene, perylene, coronene, Terylene, ovalene, quoterylene, andcircumanthracene; derivatives (such as triphenodioxazine,triphenodithiazine, hexacene-6,15-quinone) which are prepared bysubstituting some of carbon atoms of polyacens with atoms such as N, S,and O, or a functional group such as a carbonyl group; polymers such aspolyvinylcarbazoles, polyphenylenesulfide, and polyvinylenesulfide. Ofparticular interest in representative embodiments are polypyrrole,polythiophene, polyaniline or their derivatives. In representativeembodiments, the polymer is a water-soluble conducting polymer. Incertain of these embodiments, the water-soluble conducting polymer is asubstituted organic conducting polymer, where the polymer comprises anionizable group or groups. By ionizable group is meant a moiety that, atan appropriate pH is capable of carrying a net positive or negativecharge. Ionizable groups of interest include, but are not limited to:carboxyl groups, amino groups, etc. In certain embodiments, thewater-soluble conducting polymer is a substituted polyaniline, such as apoly aniline substituted with ionizable groups, e.g., carboxyl groups,such as poly(aniline-2-carboxylic acid).

In the subject colloids, metal particles are surface coated with aconducting polymer and suspended in a liquid medium, typically anaqueous medium. By “surface coated” is meant that at least a portion ofthe surface of the particles, if not the entire surface of theparticles, is covered with a layer of conducting polymer molecules. Inrepresentative embodiments, the layer or coating of conducting polymeris a monolayer, such that a single layer of polymer molecules covers thesurface of the particles.

The dimensions of the particles may vary, but in representativeembodiments range from about 1 nm to about 1 micrometer, such as fromabout 1 nm to about 100 nm, including from about 30 nm to about 60 nm.In representative embodiments, the particles have a narrow particle sizedistribution. By narrow particle size distribution is meant that thestandard deviation of the particles does not exceed about 30%, and incertain representative embodiments does not exceed about 20%, e.g., doesnot exceed about 17%, including does not exceed about 10% of the averagediameter.

With respect to the polymer component of the subject composites, thepolymer component has an average molecular weight ranging from about1,500 Da to about 32,000 Da, such as from about 5,000 Da to about 7,000Da, including from about 23,000 Da to about 27,000 Da. The polymercomponent is further characterized by having a narrow size dispersity,such that at least about 45 number %, particularly at least about 25number % of the polymer molecules adsorbed to the surface of theparticles have a molecular weight that is at least about 55%, such as atleast about 75% of the average molecular weight of the all of themolecules absorbed to the surface.

Because of the above features regarding narrow size distribution andnarrow size dispersity, the colloids are homogenous or uniform withrespect to the polymer-coated particles thereof.

The density of the the colloids may vary, but in representativeembodiments ranges is at least about 1.01, such as at least about 1.05,and may be as high as 1.30 or higher, where the density may range fromabout 1.07 to about 1.10, such as from about 1.085 to about 1.095, ascompared to the density of water at 20° C.

The concentration of particles in the liquid medium in the subjectcolloids may vary, but ranges in certain embodiments from about 1×10¹⁰to about 1×10¹⁵ particles/ml, such as from about 1×10¹¹ to about 5×10¹¹particles/ml, including from about 2×10¹¹ to about 3.75×10¹¹ particlesper ml.

In certain embodiments, the metal and conductive polymer components arematched with respect to an optical parameter, such as absorbance. Inrepresentative embodiments, the metal and conductive polymer componentsare ones that, when measured separately using the protocol describedbelow in the experimental section below, have an absorbance maximum thatdiffers by less than about 50 nm, such as by less than about 40 nm,including by less than about 25 nm. The “common” absorbance maximum(i.e., the average of the two individual absorbance maxima) may vary,ranging from about 1 to about 10, such as from about 3 to about 4.Representative matched metal/conductive polymer pairings of interestinclude, but are not limited to: gold/polyanilines (e.g.,gold/poly(aniline-2-carboxylic acid); silver/poly(thiophene-3-carboxylicacid); and the like.

In certain embodiments, the composite colloid is more sensitive tochanges in refractive index of the liquid medium in which the particlesare suspended as compared to a control colloid that is made up of metalparticles not coated with the conductive polymer. By more sensitive ismeant at least about 10 fold more sensitive, such as at least about 100fold more sensitive, including at least about 1,000 fold more sensitive,compared to a control, as determined using the assay reported in theExperimental Section, below.

In certain embodiments, the particles display a ligand, e.g., thatspecifically binds to an analyte of interest, a therapeutic moiety,etc., on their surface. By display is meant that the ligand isimmobilized on the surface of the particle, where the ligand may becovalently or non-covalently bound to the surface of the particle. Thedensity of ligand on the particle surface may vary, but may range fromabout 2 to about 50, such as from about 5 to about 25 molecules perparticle.

As indicated above, a variety of different types of ligands may bedisplayed on the surface of the particles of the colloids. In certainembodiments, the particular ligand that is present depends on the natureof the analyte that is to be bound by the ligand in a given application,such as the analyte detection applications discussed below.Representative ligands of interest include, but are not limited theligands discussed above, such as nucleic acids, peptides, etc.

The pH of the colloid may vary, and in representative embodiments rangesfrom about 2 to about 12, such as from about 4.5 to about 9.0. Thecolloids may include a number of different additional components apartfrom the polymer coated metal particles, where additional components ofinterest include, but are not limited to: salts, buffering agents,detergents, stabilizers, and the like.

In certain representative embodiments, the colloid is substantially freeof non-adsorbed polymer, i.e., the liquid component of the colloid haslittle, if any, free polymer present therein. As such, the concentrationof free polymer in solution in the liquid medium of the colloid, ifpresent at all, does not exceed about 5%, and more particularly does notexceed about 1% of the quantity used.

Methods of Fabrication

The subject colloids may be prepare using any convenient protocol thatresults in the production of a colloid of the invention, e.g., asdescribed above. In a representative embodiment, an initial or precursormetal colloid and a water-soluble conductive polymer are combined witheach other in a manner sufficient for the water-soluble conductivepolymer to adsorb to the surface of particles of the metal colloid andthereby produce the product composite colloid of the invention.

In representative embodiments, a first volume of a metal colloid iscombined with a second volume of a solution of the water-solublepolymer. The ratio of the volumes of colloid to polymer solution mayvary, but in certain embodiments ranges from about 100 to about 1, suchas from about 50 to about 20, including from about 10 to about 5. Incertain embodiments, the colloid and solution polymer are combined byintroducing the volume of colloid into the polymer solution. In otherembodiments, the colloid and solution polymer are combined byintroducing the volume of polymer solution into the colloid. In certainembodiments, combination of the volumes occurs with agitation, e.g., bystirring one of the fluids while the other is added to it, by combiningthe volumes while moving, e.g., shaking, the container in which they arecombined, etc.

The metal colloid that is combined with the water-soluble polymer is, inrepresentative embodiments, a metal colloid of a noble metal suspendedin an aqueous liquid medium. In representative embodiments, the colloidis uniform with respect to the nature of the metal particles, where theparticles have an average diameter ranging from about 2 nm to about 1μm, such as from about 3 nm to about 60 nm, including from about 5 nm toabout 30 nm and a narrow size distribution, as described above. Inrepresentative embodiments, the density of the particles in the mediumranges from about 1.01 to about 1.30, such as from about 1.02 to about1.10. In certain embodiments, the pH of the colloid is chosen to ensurethat the metal particles of the colloid have a negatively chargedsurface, where the pH may range from about 2 to about 12, including fromabout 1 to about 10, such as from about 3 to about 5.

The water-soluble polymer solution is, in representative embodiments, asolution of a water soluble conducting polymer, as described above,where the concentration of polymer in the solution may range from about0.02 to about 2 g/100 ml, such as from about 0.02 to about 0.5 g/100 ml,including from about 0.2 to about 0.3 g/100 ml. The average molecularweight of the polymer ranges, in representative embodiments, from about1,500 Da to about 32,000 Da, such as from about 5,000 Da to about 7,000Da, and has a narrow size dispersity, where at least about 55%, such asat least about 75% of the polymers present in the solution have amolecular weight that is at least about 90 to about 110%, such as atleast 95 to about 105% of the average molecular weight. In certainembodiments, he pH of the water-soluble polymer solution is chosen sothat the water-soluble polymers are positively charged, where the pH mayrange in representative embodiments from about 2 to about 7, such asfrom about 3 to about 5.

In certain embodiments, the volumes of the metal colloid and watersoluble polymer solution, as well as parameters thereof (e.g., density,pH, concentration, etc.) that are combined in this step of the subjectinvention are ones that have been predetermined to result in theproduction the product composite colloid that is stable andsubstantially free of solution phase polymer, where by “substantiallyfree is meant that the concentration of solution phase polymer is lessthan about 5%, such as less than about 1%. A feature of theseembodiments is that this product colloid is produced without any washingor other step that removes solution phase polymer from the colloid. Theappropriate volumes and parameters thereof for practicing theseembodiments of the subject methods may be determined using the protocolsdiscussed in the experimental section, below.

In combining the metal colloid and the water-soluble polymer, the twocomponents are combined into a reaction mixture, and the resultantreaction mixture maintained for a period of time sufficient for thedesired colloid to be produced. Generally, the reaction mixture ismaintained at a temperature ranging from about 15° C. to about 30° C.,e.g., from about 18° C. to about 22° C., for a period of time rangingfrom about 5 minutes to about 60 minutes, such as from about 10 minutesto about 20 minutes.

In certain embodiments, the methods may further include a step ofmodifying the surface of the composite particles of the colloid todisplay a ligand, e.g., that specifically binds to an analyte ofinterest. Where desired, a ligand may be immobilized directly orindirectly, e.g., via a linking group, on a surface of the particlesusing any convenient protocol, including one that employs covalentbonding or non-covalent bonding of the ligand to the particle, e.g.,either to the metal component directly or to the polymer present on asurface of the particle, e.g., via reaction with a functional grouppresent on the polymer. The ligand may be any of a number of differenttypes of molecules, e.g., nucleic acid, peptide, organic and inroganicsmall molecules, etc., as reviewed above. As indicated above, wheredesired the reaction mixture may be agitated.

Utility

The subject colloids, as described above, may be employed in a varietyof different applications. For example, the subject colloids may be usedto screen a sample for the presence or absence of one or more targetanalytes in the sample. As such, the invention provides methods ofdetecting the presence of one or more target analytes in a sample.

In such applications, a volume of colloid, e.g., one that includes aligand specific for the analyte, is contacted with the sample to bescreened, and an optical parameter of the colloid is monitored to detecta change therein, e.g., a change in absorption of the colloid at a givenwavelength. Any convenient optical parameter may be assessed ormonitored in this step, where representative parameters include, but arenot limited to: absorption, scattering, fluorescence, luminescence andthe like. The optical parameter may be monitored using any convenientdevice and protocol, where suitable protocols are well known to those inthe art and representative protocols are described in greater detail inthe experimental section, below. The presence or absence of a change inthe optical parameter is then used to make a determination of whether ornot the analyte of interest is present in the sample.

In the broadest sense, the methods may be qualitative or quantitative.As such, where detection is qualitative, the methods provide a readingor evaluation, e.g., assessment, of whether or not the target analyte ispresent in the sample being assayed. In yet other embodiments, themethods provide a quantitative detection of whether the target analyteis present in the sample being assayed, i.e., an evaluation orassessment of the actual amount of the target analyte in the samplebeing assayed. In such embodiments, the quantitative detection may beabsolute or, if the method is a method of detecting two or moredifferent target analytes in a sample, relative. As such, the term“quantifying” when used in the context of quantifying a targetanalyte(s) in a sample can refer to absolute or to relativequantification. Absolute quantification may be accomplished by inclusionof known concentration(s) of one or more control analytes andreferencing the detected level of the target analyte with the knowncontrol analytes (e.g., through generation of a standard curve).Alternatively, relative quantification can be accomplished by comparisonof detected levels or amounts between two or more different targetanalytes to provide a relative quantification of each of the two or moredifferent analytes, e.g., relative to each other.

The subject methods can be employed to detect the presence of one ormore target analytes in a variety of different types of samples,including complex samples having large amounts of non-target entities,where the subject methods provide for detection of the targetanalytes(s) with high sensitivity. As such, the subject methods arehighly sensitive methods of detecting one or more target analytes in asimple or complex sample. The sample that is assayed in the subjectmethods is, in certain embodiments, from a physiological source. Thephysiological source may be eukaryotic or prokaryotic, withphysiological sources of interest including sources derived from singlecelled organisms such as bacteria and yeast and multicellular organisms,including plants and animals, particularly mammals, where thephysiological sources from multicellular organisms may be derived fromparticular organs or tissues of the multicellular organism, or fromisolated cells or subcellular/extracellular fractions derived therefrom.

The methods of the present invention may be used to detect a widevariety of analytes. Analytes of interest may be present as liquids,solids or gases (eg., organophosphates, etc.). Analytes of interest canbe a proteinacious molecules, such as, but not limited to, proteinaciousanalytes, including peptides and proteins and fragments thereof, as wellas prions and other proteinaceous types of analytes, where the analytesmay be a single molecule, a complex that includes two or more molecularsubunits, which may or may not be covalently bound to each other, amicroorganism, e.g., virus or single celled pathogen, a cell, amulticellular organism or portion thereof, and the like.

In addition, the subject methods may also be used to screen forcompounds that modulate the interaction of a given specific bindingmember pair. The term modulating includes both decreasing (e.g.,inhibiting) and enhancing the interaction between the two molecules. Forexample, where the colloid displays a first member of a binding pair andthe colloid is contacted with the second member in the presence of acandidate agent, the effect of the candidate agent on the interaction ofthe binding member pairs can be evaluated or assessed.

A variety of different candidate agents may be screened by the abovemethods. Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500Dolton. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides and oligopeptides. Alternatively, libraries of naturalcompounds in the form of bacterial, fungal, plant and animal extractsare available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Agents identified in the above screening assays find use in a variety ofmethods, including methods of modulating the activity of the targetanalyte, and conditions related to the presence and/or activity thereof.

Additional applications of the subject colloids include therapeuticapplications, e.g., as drug delivery vehicles. For example, atherapeutic agent can be displayed on a surface of the nanoparticles,and an effective amount of the colloid that is made up of thenanoparticles administered to a subject to treat the subject. Wheredesired, nanoparticles of the colloid may be further modified to includea targeting moiety, e.g., to direct the nanoparticles to a desiredlocation.

Kits & Systems

As summarized above, also provided are kits and systems for use inpracticing the subject methods. The kits and systems at least includethe subject colloids or components thereof, as described above. The kitsand systems may also include a number of optional components that finduse in the subject methods. Optional components of interest includebuffers, and the like.

In certain embodiments of the subject kits, the kits will furtherinclude instructions for practicing the subject methods or means forobtaining the same (e.g., a website URL directing the user to a webpagewhich provides the instructions), where these instructions are typicallyprinted on a substrate, which substrate may be one or more of: a packageinsert, the packaging, reagent containers and the like. In the subjectkits, the one or more components are present in the same or differentcontainers, as may be convenient or desirable.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

The following describes a process for the production ofpoly(aniline)-colloidal gold nanoparticles and reports theopto-electronic characterization of the material synthesized.

I. Synthesis

A. Synthesis of Water-Soluble Poly(aniline-2-carboxylic Acid)(PANI-COOH)

The synthesis consists in oxidizing the monomer in aqueous solution withiron chloride and the detailed procedure has been described previously(Englebienne, P., Weiland, M. Water-soluble conductive polymerhomogeneous immunoassay (SOPHIA), a novel immunoassay capable ofautomation. J Immunol. Methods 1996, 191, 159-170; Englebienne, P.,Weiland, M. Synthesis of water-soluble carboxylic and aceticacid-substituted poly(thiophenes) and application of their photochemicalproperties in homogeneous competitive immunoassays. Chem. Commun., 1996,1651-1652; Englebienne, P., Indicator reagents for the detection ordosage of an analyte, kits containing them and detection or dosageprocedures. Eur. Pat. 0623 822, 1994). Poly(thiophenes) can also beobtained by the same procedure, but the oxidation uses ammoniumperoxydisulfate in addition to the iron chloride. At the end of thesynthesis, the pH is raised up to 12 with NaOH pellets in order toprecipitate the iron chloride as the hydroxyde and to decompose theperoxydisulfate. Iron hydroxyde is removed by filtration and the polymersolution is used as such. In the present example, the synthetic yieldwas improved by two means: improved solubilization of the monomer indimethylformamide:water (1:10) and polymerization under stirring usingheat and reflux.

As the gel permeation (GP) chromatographic profile shown in FIG. 1indicates, the solutions of the polymeric poly(thiophene-3-carboxylicacid) and of PANI-COOH are quite homogeneous with major peaks at a sizeof approx. 18,000 (20.5 min.) and 6,000 Dolton (24 min.), respectivelyand minor peaks with a size of approx. 25,000 (19 min.) and 15,000Dolton (21 min.), respectively. Such sizes correspond to oligomers madeof 45 and 110 repeats for PANI-COOH, respectively. Please note thatthese values are not absolute values because the column has beencalibrated with globular proteins and the polymers are not necessarilyglobular in shape.

-   Chromatographic conditions:-   HPLC column: TSK 2000 SW, 7.5×600 mm.-   Elution: 50 mM phosphate pH 7.4.-   Flow rate: 1 ml/min.-   Injection: 50 μl.-   Detection: O.D. 254 nm, 0.5 AUFS.-   Paper speed: 1 mm/min    B. Synthesis of Colloidal Gold Nanoparticles.

The nanoparticles are obtained by reduction of a boiling hydrogentetrachloroaurate solution by sodium citrate. The process is well-knownand is described in the following publication (Englebienne, P., VanHoonacker, A., Verhas, M. High throughput screening using the surfaceplasmon resonance effect of colloidal gold. Analyst, 2001, 126,1645-1651). Detailed procedures are provided in the book referenced toabove to obtain nanoparticles of various sizes. The nanoparticles usedin the present report have an approximate diameter of 50 nm and arehomeodisperse.

C. Synthesis of Composite Poly(aniline-2-carboxylate)-Colloidal GoldNanoparticles.

Colloidal noble metal nanoparticles are negatively charged over a widerange of pH. Procedures designed for coating such nanoparticles withproteins take advantage of this property. The nanoparticles are mixedwith the protein at a pH below the protein pl and the protein adsorbs onthe particle surface by charge interaction. Such protein-coatedparticles are stable and do not flocculate in the presence of high saltconcentrations. Common procedures involve the addition of proteins inexcess to the gold so as to avoid the formation of bridges betweenindividual nanoparticles, made by protein molecules. After proteinadsorption, the nanoparticles are centrifuged and washed so as to removethe excess protein. The colloid is then resuspended in a suitablebuffer. During the previous years, we have developed a process thatsimplifies tremendously that procedure, which is described in our bookand our J. Mater. Chem. Publication referenced to above. The principleconsists in mixing the gold colloid in test tubes with increasingprotein concentrations at a suitable pH. After mixing, a 1 M NaClsolution is added. In tubes were the particles are not completelystabilized with a protein layer, the nanoparticles flocculate, whichinduces a strong red-shift in their visible absorption spectrum, fromthe native surface plasmon resonnance (SPR) peak of 520 nm (for gold) upto 600-800 nm, resulting in a decrease in O.D. at the SPR peak. Abinding isotherm constructed from the spectrum data and proteinconcentrations added to the gold colloid therefore allows to determinethe minimal protein concentration required to fully stabilize the goldsol, in other words, the protein concentration required for coatingsingle nanoparticles with a complete protein layer. The process is thenscaled-up for the production of larger volumes.

In the present case, we reasoned that a water-soluble conductive polymersubstituted by ionizable groups along the backbone could behave exactlyin the same way. For PANI-COOH for instance, we considered that at lowpH, all the carboxylic groups would be protonated and would therefore becapable of coating colloidal gold nanoparticles by charge adsorption,producing stable colloidal composites. In order to test the hypothesis,we first examined the effect of the pH to the possible capacity ofPANI-COOH to stabilize colloidal gold nanoparticles.

The protocol used was as follows. To a series of plastic tubescontaining 0.01 ml of the PANI-COOH solution, 1 ml of the colloidal goldsolution was rapidly injected with a positive displacement pipette andthe tube immediately vortex-mixed. The colloidal gold solution added toeach independent tube was adjusted to various. pH values using either 10mM sodium carbonate or 300 mM HCl solutions, respectively. Then, 0.5 mlof a 1 M solution of sodium chloride was added to each tube which wasfurther vortex mixed, so as to flocculate the particles that were notstabilized. UV-vis. spectra were recorded for each solution. The peakwavelength and absorbancy at the maximal (SPR) wavelength were recordedand plotted versus pH. The results are shown in the FIG. 2.

As shown on FIG. 2, the SPR peak wavelength of the gold colloid shiftsprogressively to the red from 550 to 660 nm from pH 2 to 5 and thendrops rapidly back to 560 nm above pH 5. The O.D. at the peak shows amaximum around pH 3 and falls rapidly to the half above pH 5. The lowO.D. at the SPR peak above pH 5 indicates that the flocculation is suchthat most of the particles precipitate at the bottom of the cuvette. Thestable maximal wavelength and O.D. observed correspond to the solublePANI-COOH remaining in the supernatant. Below pH 5, the particles arelikely to be stabilized with an optimum close to pH 3. This firstexperiment led us to conclude that water-soluble PANI-COOH couldstabilize the gold nanoparticles, even if the concentration was notoptimal. Interestingly enough, these results are moreover in line withthe ionization data available for the monomer which displays two pKavalues of respectively 2.10 and 4.94 (Handbook of Chemistry and Physics,CRC Press). Thus, around the first pKa value, the molecules isprotonated and capable of stabilizing the gold particles by chargeadsorption, although above the second pKa value, the repulsion due tonegative charges of both gold and PANI-COOH allows the salt added tobridge and flocculate the particles.

Once the optimal pH for stabilizing the particles determined, it wasnecessary to determine the minimal PANI-COOH concentration required forstabilizing the particles. For this optimization, the following protocolwas used. Increasing volumes of the PANI-COOH solution (from 0.001 up to0.1 ml) were added to individual plastic tubes. The volume was adjustedto 0.1 ml in each tube by the addition of appropriate volumes ofdistilled water. After miximg, 1 ml of colloidal gold solution of whichthe pH was adjusted to 2.38 using HCl 300 mM, was rapidly injected ineach individual tube using a positive displacement pipette and each tubewas immediately vortex mixed. Then, 0.5 ml of a 1 M solution of sodiumchloride was added to each tube which was further vortex mixed, so as toflocculate the particles that were not stabilized. UV-vis. spectra wererecorded for each solution. The maximal wavelength of absorbance wasrecorded, along with the optical density (O.D.) at 750 nm which isrepresentative of the flocculation of the colloid. Results are displayedin the graph of (FIG. 3.). The maximal wavelengths of absorption of thegold and PANI-COOH solutions are respectively shown by down and up opentriangles on the FIG.

The data shown in FIG. 3. indicate that the gold particles werestabilized after incubation with 0.01-0.02 ml of PANI-COOH per ml of sol(minimal wavelength and O.D. 750 nm). These conclusions are furtherconfirmed by the data shown in FIG. 4. Here, the O.D. at the SPR peak isplotted, along with the peak wavelength versus the log volume ofPANI-COOH solution added. Because increasing volumes of PANI-COOH wereadded to the gold and given the fact that PANI-COOH absorbs at awavelength close to that of the gold colloid (560 nm), the contributionof PANI-COOH in the O.D. at the SPR wavelength for each volume wassubtratced from the data plotted. As shown by the figure, the maximalO.D. at the SPR peak of gold is observed for PANI-COOH volumes added of0.01-0.02 ml.

With these optimization data in hand, the procedure was scaled-up tohigher volumes. A batch of composite material was successfully producedas follows. The PANI-COOH solution synthesized as described above (1.5ml) was diluted with 8.5 ml distilled water in a beaker containing amagnetic stirrer bar. In a separate vessel, 100 ml of colloidal goldsolution synthesized as described above was adjusted under magneticstirring to pH 2.94 using HCl 300 mM. The PANI-COOH solution was placedon the magnetic stirrer and was mixed at the highest speed. ThepH-adjusted gold solution was then added rapidly to the PANI-COOHsolution. After mixing, the pH of the mixture was 4.67. The compositesol was further stabilized by the addition of 0.5% Tween 20. The batchwas then divided into two equal parts, the first one being buffered with50 mM sodium acetate at pH 4.95, the other one being buffered with 50 mMsodium borate at pH 9. It is important to note that reversing the orderof reagent mixing in the procedure works also.

II. Physico-Chemical Characterization of the Material

A. Preliminary Note.

Our interest in preparing a composite material made of a conductingpolymer and colloidal nanoparticles of noble metal was primarilydirected to the possible modifications in the opto-electronic propertiesof both materials taken individually. The sensitivity of these materialsto various changes in their physical environment is transduced bychanges in their electronic spectra. Consequently, in order to be ableto observe consistant changes in such spectra with the compositematerials considered, it was quite reasonable to consider nativematerials, which, taken individually in their native state, presentedsimilar energies of light absorption. This is the reason why we selectedcolloidal gold and PANI-COOH. The visible spectrum of gold nanocolloidswith particles of 50 nm of diameter suspended in water presents alocalized SPR absorption band at 520 nm (see FIG. 5). Consistantly, thevisible spectrum of a solution of PANI-COOH in water presents anabsorption band at 560 nm. The visible spectra of the original gold,PANI-COOH and of the composite material obtained by their combinationare presented in FIG. 5. As expected, the characteristics of thecomposite material spectrum result from the addition of those of theoriginal materials used for its production.

B. Stability.

FIG. 6. compares the visible absorption spectra of the compositematerial taken after synthesis and more than 3 months later. The maximalabsorption wavelength is identical and no significant loss of absorbanceat this wavelength can be observed. The composite is likely to havematured during storage as the absorption peak is more homogeneous in thespectrum taken at the later date.

Additionally, over a period of more than 3 months after the production,no sedimentation has been observed. The composite material, whether atacidic or basic pH is a stable colloidal solution. In that regard, itappears to the naked eye essentially indistinguishable from controlssolutions of colloidal gold and PANI-COOH compared under the sameconditions of concentration of materials and pH.

C. Reactivity Toward Oxido-Reduction.

C.1. Sensitivity to pH.

It is well known that conducting polymers are sensitive to pH changes.This is particularly true for poly(aniline) which changes its electronicstructure with pH. The progressive p-doping (oxidation) by protonationof the non-conducting leucoemeraldine into the conducting emeraldineresults in a shift from the absorption of high energy photons (343 nm,3.61 eV) to the absorption of both higher (330 nm, 3.75 eV) and lower(637 nm, 1.94 eV) photons. Further oxidation leads to the fullyquinonoid form pernigraniline which is insulating. A solution ofPANI-COOH behaves similarly towards light when progressively protonated(see e.g. Englebienne, P. Synthetic materials capable of reportingbiomolecular recognition events by chromic transition. J. Mater. Chem.,1999, 9, 1043-1054). Therefore, it was interesting in the first instanceto verify if the composite material was still capable of reporting thestructural changes occuring in the structure of the polymer byprotonation by similar changes in its UV-vis. spectrum. To this aim, wecompared the electronic spectra of the composite buffered at pH 4.95with the solution buffered at pH 9. For comparison, we prepared bufferedsolutions of PANI-COOH at the concentration used to prepare thecomposite and we adjusted the pH at the same respective values. Whencomparing the spectra of the two materials at the respective pHs, weobserved indeed the expected changes. The shifts in wavelength wereidentical for the conducting polymer and the composite, and theimportance of the changes in absorbance were similar in both thecomposite and plain polymer. In order to illustrate these changes, werecorded the difference spectrum between the materials adjusted at pH4.95 and the materials at pH 9 (reference cell). These differencespectra are presented in FIG. 7. The appearance of the new bands in theprotonated materials at both high (380 and 420 nm) and low (680 nm)energy photons are identical for the composite (solid line) and thenative conducting polymer (dashed line). The negative peak at 580 nm inthe polymer is shifted to shorter wavelengths in the composite i.e. inthe region of the SPR peak of gold. This results probably from changesin the energy of the conduction electrons at the metal surface.

C.2. Response to Reduction.

Another interesting feature of water soluble PANI-COOH, which is alsoshared with insoluble poly(aniline), is the photonic sensitivity of thematerial to oxidoreuction. Both protonation and changes in oxidationstates of the emeraldine salts give rise to marked transitions in theoptical spectrum (Grummt, U.-W., et al. Anal Chim. Acta 1997, 547, 253).That property was recently applied in a highly sensitive assay forascorbic acide, using a poly(aniline) film desposited in microtiterplates (Bosi, A., et al., Anal. Chem. 2000, 72, 4296). When compared tocurrently available analytcial techniques, this new assay presentsseveral marked advantages because it uses smaller sample volumes,displays a lower detection limit, and proves reproducible in an extendedrange of analyte concentrations.

In order to compare the redox sensitivity of the composite nanomaterialto the of PANI-COOH, we incubated at different pH values both materialswith increasing concentrations of ascorbic acid (0.9-500 mg/l) andrecorded the difference spectra versus the mixture devoid of reductant.Whatever the pH, both materials reported their reduction by aprogressive decrease in absorbance at 600 nm. In agreement withpreviously reported results for the microtiter plate assay, we observedthe strongest changes in the 600 nm absorbance intensity for bothmaterials at pH values below 4. The composite nanomaterial displayed,however, a stronger optical reactivity to reduction than water-solublePANI-COOH.

With an aim to further document the difference, we designed a simpleassay for ascorbic acid in solution based on the previously describedprocedure, using the reagents in aqueous solution rather than as a solidfilm. In the presence of ascorbic acid, the reproducibility of theoptical measurements was rather poor with PANI-COOH, as can be see bythe error bars for that dose-response curve, shown in FIG. 8 (uppercurve). This is most likely due to the progressive loss of solubility ofPANI-COOH at low pH values. In contrast, the optical response of thecomposite nanomaterial to reduction with ascorbic acid was highlyreproducible and the dose-response curve was must steeper, as shown inFIG. 8 (lower curve). With PANI-COOH, the absorbance change for a 20mg.l ascorbic acid dose (0.076 AU) was comparable to that observed inthe microtiter plate assay (0.118 AU) [20]. For the same analyte dose,the response for the composite nanomaterial was, however, much higher(0.22 AU). We further compared the least detectable doses (LDD), whichwere measure at three times the standard deviation of the zero responseof each system. The calculated LDD was 3.39 mg/l for the PANI-COOHassay, which was close to the detection limit of the previously reportedmicrotiter plate assay. Comparitively, the composite nanomaterial was 60times more sensitive, with a LDD calculated at 0.057 mg/l.

Such high sensitivity with good reproducibility and linearity insolution makes the composite nanomaterial an excellent biosensingreagent requiring a level of redox sensitivity at the lower micromolarrange. Some examples of applications in which the composite nanomaterialfinds use include the plasmatic antioxidant capacity and the detectionand quantitation of superoxide radicals in cells.

D. Reactivity Towards the Refractive Index of the Medium.

The localized SPR peak of a colloidal gold nanoparticle solution isexquisitely sensitive to changes in the refractive index of the mediumthat surround them. The sensitivity is such that we use this property tomeasure biomolecular interactions occuring at the surface of particlescoated with antibodies or receptors (Englebienne, P., Van Hoonacker, A.,Verhas, M. Surface plasmon resonance: Principles, methods andapplications in the biomedical sciences. Spectroscopy, 2003, 17,255-273; Englebienne, P., Van Hoonacker, A., Verhas, M., Khlebtsov, N.G. Advances in high throughput screening: Biomolecular interactionmonitoring in real-time with colloidal metal nanoparticles Combin. Chem.High-Throughput Screen., 2003, 6, 777-787).

We use to test the reactivity of materials toward changes in therefractive index of the medium with glycerol. The colloidal solution(0.5 mL) is mixed with 1.5 mL of aqueous solutions containingrespectively 0, 10, 20, 33.3, 50. 66.6 and 100% of glycerol. The finalconcentrations of glycerol in the medium are thus 0, 7.5,15, 25, 37.5,50 and 75%, respectively, which changes the refractive index of themedium from that of water (1.3326) to progressively and respectively1.3418, 1.3508, 1.3637, 1.3806, 1.3968 and 1.4355. When goldnanoparticles are subjected to such a treatment, the localised SPR peakaround 520 nm decreases slightly and the peak is shifted progressivelyto longer wavelengths, from 550 up to 700 nm. This is exemplified inFIG. 9, which shows the difference spectra of the gold sol in aqueoussolutions containing 7.5, 25, 37.5, 50 and 75% of glycerol versus thesame colloid in water.

Because in the composite, the gold nanoparticles are covered with alayer of conductive polymer (PANI-COOH), the opto-electronic behavior ofthis new material toward the refractive index surrounding thenanoparticles could be modified, or even completely supressed.Therefore, it was important to check this behavior. The reactivity ofthe PANI-COOH-gold composite nanoparticles buffered at pH 9 towardchanges in refractive index of the medium is exemplified by thedifference spectra shown in FIG. 10.

Interestingly, the progressive localized SPR wavelength shift towardlonger wavelengths occurs for low glycerol concentrations, but at higherconcentrations, it decreases progressively, whilst a sharp and intensepeak appears at a much higher photonic energy excitation (350 nm, 3.54eV). The variation of absorbance at the typical wavelengths as afunction of the changes in refractive index of the medium are shown inFIG. 11.

Typically, the peaks at low energy wavelengths progressively shift tohigher energy wavelengths with a consistent and continuous increase inabsorbance at 350 nm. Neither gold nanocolloids, nor PANI-COOH solutionspresent such a behavior in presence of increasing concentrations ofglycerol. This new behavior of the composite material most probablyresults from the conjunction of the high energy electrons of the polymerwith the conduction electrons at the gold nanoparticle surface. Thiscreates a cloud of collectively oscillating electrons at the interfacewith higher energy than that of the conduction electrons of gold. Thisphenomenon creates a new SPR band in the composite material at shorterwavelengths which absorbs high energy photons to create higher energyphoton-plasmon evanescent waves than those that occur in the native goldnanoparticles.

Interestingly, a similar phenomenon has been observed withantibody-coated nanoparticles of gold and silver plated as a monolayeron quartz plates, upon binding of the protein antigen (Frederix, F.,Friedt, J. M., Choi, K. H., Laureyn, M., Campitelli, A., Mondelaers, D.,Maes, G., Borghs, G. Biosensing based on light absorption of nanoscaledgold and silver nanoparticles. Anal. Chem., 2003, 75, 6894-6900).

As a consequence of this changed opto-electronic behavior, themeasurement of absorbance changes at 350 nm for the composite materialis approximately 4 times more sensitive to changes in the refractiveindex of the medium than the same absorbance changes at 575 nm for goldnanoparticles. This is exemplified in FIG. 12.

It is evident from the above discussion and results that the subjectinvention provides new materials and methods that are useful in avariety of applications.

The preparation of the composite material consists of mixing thewater-soluble conducting polymer at a fixed concentration with the metalnanoparticles at a suitable pH. The composite nanomaterial is stable formonths at room temperature in colloidal buffered solutions. Theopto-electronic properties of the new composite nanomaterial aresensitive to oxido-reduction and changes in the refractive index of itssurrounding medium and their sensitivity is enhanced when compared tothe sensitivity of gold nanoparticles and conducting polymer takenseparately. The composite nanomaterial presents new opto-electronicproperties when compared to those of the separate materials of which itis composed.

As such, use of the subject colloids in analyte detection applicationsoffers an advantage over the sensitive fluorescent dyes or particles incurrent use because neither conductive polymers nor colloidal metalssuffer from photobleaching (i.e., the irreversible photochemicalprocesses leading to non-fluorescent products) or “blinking” effects(i.e. intermittent signal emission due to photoionization) that limittheir performance as reagents. The detection systems of the presentinvention find use in many commercial products including homogeneous(competitive or sandwich) immunoassays, high throughput screening,protein arrays, PCR product detection and quantitation in real-time andhybridization detection, to name a few representative applications. Assuch, the subject invention represents a significant contribution to theart. Although the foregoing invention has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope.

Furthermore, all examples and conditional language recited herein areprincipally intended to aid the reader in understanding the principlesof the invention and the concepts contributed by the inventors tofurthering the art, and are to be construed as being without limitationto such specifically recited examples and conditions. Moreover, allstatements herein reciting principles, aspects, and embodiments of theinvention as well as specific examples thereof, are intended toencompass both structural and functional equivalents thereof.Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of present invention is embodied bythe appended claims.

1. A method of producing a metal/conductive polymer composite colloid,said method comprising: combining: (i) a metal colloid; and (ii) awater-soluble conductive polymer; under conditions sufficient for saidwater-soluble conductive polymer to adsorb to metal particles of saidcolloid to produce a metal/conductive polymer composite colloid.
 2. Themethod according to claim 1, wherein said metal colloid and saidwater-soluble conductive polymer are combined with agitation.
 3. Themethod according to claim 2, wherein said metal colloid and saidwater-soluble conductive polymer are combined by combining first volumeof said metal colloid a second aqueous volume of said water-solubleconductive polymer.
 4. The method according to claim 3, wherein saidmetal colloid and said water-soluble conductive polymer are combined byintroducing a said first volume into said second volume while saidsecond volume is agitated.
 5. The method according to claim 3, whereinsaid metal colloid and said water-soluble conductive polymer arecombined by introducing a said second volume into said first volumewhile said first volume is agitated.
 6. The method according to claim 1,wherein metal particles of said metal colloid and said water-solubleconductive polymer are oppositely charged.
 7. The method according toclaim 6, wherein said metal particles are negatively charged.
 8. Themethod according to claim 6, wherein said metal colloid and saidwater-soluble conductive polymer are combined by combining first volumeof said metal colloid a second aqueous volume of said water-solubleconductive polymer.
 9. The method according to claim 8, wherein saidfirst and second volumes have a respective pH that is chosen so thatsaid metal particles and water-soluble polymer are oppositely charged.10. The method according to claim 3, wherein said second volume has awater-soluble conductive polymer concentration that provides forsubstantially no free water-soluble polymer in said metal/conductivepolymer composite colloid.
 11. The method according to claim 1, whereinsaid method further comprises modifying surfaces of particles of saidmetal/conductive polymer composite colloid to display a ligand. 12.-17.(canceled)
 18. The method according to claim 1, wherein said metalcolloid is a colloid of a noble metal.
 19. The method according to claim18, wherein said noble metal is chosen from gold and silver.
 20. Themethod according to claim 19, wherein said noble metal is gold.
 21. Themethod according to claim 1, wherein said water-soluble conductivepolymer is an organic polymer.
 22. The method according to claim 21,wherein said organic polymer comprises ionizable moieties.
 23. Themethod according to claim 22, wherein said ionizable moieties arecarboxylic acid moieties.
 24. The method according to claim 1, whereinsaid water-soluble conductive polymer is a substituted polyaniline. 25.The method according to claim 24, wherein said substituted polyanilineis poly(aniline-2-carboxylic acid).
 26. The method according to claim 1,wherein the density of said metal colloid ranges from about 1.01 toabout 1.30.
 27. The method according to claim 1, wherein said metalcolloid comprises metal particles having a diameter ranging from about 1nm to about 1 μm.
 28. The method according to claim 1, wherein saidmetal colloid is homogenous with respect to size of metal particlesthereof.
 29. The method according to claim 3, wherein the concentrationof water-soluble conductive polymer in said second volume ranges fromabout 0.02 g/100 ml to about 2 g/100 ml.
 30. The method according toclaim 29, wherein said water-soluble conductive polymer of said secondvolume has an average molecular weight ranging from about 1,500 Da toabout 32,000 Da.
 31. The method according to claim 30, wherein saidsecond volume is homogenous with respect to said water-solubleconductive polymer.
 32. A metal/conductive polymer composite colloidproduced according to the method of claim
 1. 33. A stablemetal/conductive polymer composite colloid comprising metal particlessurface coated with a conductive polymer layer and suspended in anaqueous medium. 34.-54. (canceled)
 55. A stable metal/conductive polymercomposite colloid comprising metal particles surface coated with aconductive polymer layer and suspended in an aqueous medium, where saidparticles display a ligand. 56.-74. (canceled)
 75. A method forscreening a sample for the presence of an analyte, said methodcomprising: (a) contacting said sample with a stable metal/conductivepolymer composite colloid to produce an assay mixture, wherein saidcolloid metal particles surface coated with a conductive polymer layerand displaying a ligand for said analyte; and (b) detecting an opticalparameter of said assay mixture to screen said sample for the presenceof said analyte. 76.-82. (canceled)